Sandbox77

This sandbox is in use until August 1, 2011 for UMass Chemistry 423. Others please do not edit this page. Thanks!

HIV Reverse Transcriptase
Group Members: Anh Huynh, Jordan Schleeweis, Max Moulton, Sally Stras

A. INTRODUCTION

HIV-1 reverse transcriptase is required for the creation of double-stranded DNA from mutant single-stranded RNA (hence the term "reverse transcription") [2]. Once mutant single-stranded RNA is transformed into double stranded DNA, the HIV virus can be incorporated into host cells [3]. Anti-HIV drugs frequently utilize mechanisms to inhibit HIV-1 reverse transcriptase. By preventing the proper functionality of HIV-1 reverse transcriptase, the virus's ability to replicate and expand are greatly reduced. These drugs are not always effective because reverse transcription is at high risk for error. DNA mutations due to error facilitate virus resistance to drugs [3]. As a result, anti-HIV drugs must be continuously modified.

B. OVERALL STRUCTURE

HIV-1 RT 3dlk is an asymmetric heterodimer that contains two subunits, p66 (blue) and p51 (green). The two subunits p66 and p51 consist of 560 and 440 residues respectively. Both subunits consist of Alpha Helicies and Beta Sheets alternating at random intervals. There are a total of 32 alpha helicies and 12 beta sheets in the heterodimer. The majority of the beta sheets are constructed in anti-parallel form with a few exceptions. The two subunits are not related in the sense of a similar rotational axis. There are two polymerase domains and one RNase-H domain. The two polymerase domains are the same in sequence but different in conformation. There is one ligand; a sulfate ion that binds to the p66 subunit. The two subunits come together to form the binding region for DNA/RNA. [2]

C. DRUG BINDING SITE

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs [2]. Structures of HIV-1 RT have been tested with inhibitor drugs to test the effects of inhibition on RT. These structures have to be specifically engineered in a cost effective way. HIV-1 RT is ligand-independent. The binding sites for possible drugs in HIV-1 RT are in the same location as where the DNA lines up to be replicated [3]. The most successful inhibitor that has been incorporated into HIV-1 RT is called Nevirapine, which binds with the DNA on the hydrophobic inside of the RT. Fragment screening with RT52A/TMC278 crystals is a technique used for finding potential new inhibitors and sites for inhibitors to bind [2]. These tests yielded a site for drug binding interaction called the thumb conformation which is nonengineered unliganded HIV-1 RT [2]. A drug must exhibit noncompetitive inhibition, like Nevirapine, and must bind both the primer-template and dNTP substrates. The specific binding of Nevirapine is a pocket between the beta sheets of the palm (Chain A) and the thumb (highlighted above) subdomains [3]. The drug prevents movements of this thumb subdomain which is needed for the catalytic cycle of HIV. Research continues in this area to determine other mutations in RT that can be engineered for effective drug binding.

D. ADDITIONAL FEATURES

An important part of the HIV reverse transcriptase is an alpha-turn-alpha region of the p66 subunit that has been known to interact with incoming DNA. This portion of the DNA polymerase is the helix clamp. Is it located in the 'thumb' region and is present in many nucleic acid polymerases and therefore known to assist in the nucleic acid binding [1]. The RNase H domain, located on the p66 subunit, takes part in the cleavage of the RNA/DNA intermediate. The RNase H cleaves the RNA primer from the RNA/DNA hybrid allowing a sythesized DNA to be completed [4].

References

[1] Gotte M, Hermann T, Heumann H, Meier T. The 'helix clamp' in HIV-1 reverse transcriptase: a new nucleic acid binding motif common in nucleic acid polymerases. Nucleic Acids Res.1994 Nov 11;22(22):4625-33.

[2] Bauman JD, Das K, Ho WC, Baweja M, Himmel DM, Clark AD Jr, Oren DA, Boyer PL, Hughes SH, Shatkin AJ, Arnold E. Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design. Nucleic Acids Res. 2008 Sep;36(15):5083-92. Epub 2008 Aug 1. PMID:18676450 doi:10.1093/nar/gkn464

[3] L. A. Kohlstaedt, J. Wang, J. M. Friedman, P. A. Rice and T. A. Steitz. Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor. Science. 2002 June 26: 256(5056):1783-1790.

[4] Jay F. Davies, II, Zuzana Hostomska, Zdenek Hostomsky, Steven R. Jordan and David A. Matthews. Crystal Structure of the Ribnuclease H domain of HIV-1 reverse transcriptase. Science. 1991 Apr 5;252(5002):89-95. Credits

Introduction -- Anh Huynh

Overall structure --Maxwell Moulton Drug binding site -- Jordan Schleeweis

Additional features -- Sally Stras